Calyxamides A and B, cytotoxic cyclic peptides from the marine sponge Discodermia calyx

Article abstract of DOI:10.1021/np2009187

Cyclic peptides containing 5-hydroxytryptophan and thiazole moieties were isolated from the marine sponge Discodermia calyx collected near Shikine-jima Island, Japan. The structures of calyxamides A (1) and B (2), including the absolute configurations of all amino acids, were elucidated by spectroscopic analyses and degradation experiments. The structures are similar to keramamides F and G, previously isolated from Theonella sp. The analysis of the 16S rDNA sequences obtained from the metagenomic DNA of D. calyx revealed the presence of Candidatus Entotheonella sp., an unculturable δ-proteobacterium inhabiting the Theonella genus and implicated in the biosynthesis of bioactive peptides.

Full text of DOI:10.1021/np2009187

Note
pubs.acs.org/jnp
Calyxamides A and B, Cytotoxic Cyclic Peptides from the Marine
Sponge Discodermia calyx
Miki Kimura,† Toshiyuki Wakimoto,*,† Yoko Egami,† Karen Co Tan,† Yuji Ise,‡ and Ikuro Abe*,†
†Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, Miura, Kanagawa, 238-0225, Japan
S
* Supporting Information
ABSTRACT: Cyclic peptides containing 5-hydroxytryptophan and thiazole moieties were isolated from the marine sponge
Discodermia calyx collected near Shikine-jima Island, Japan. The structures of calyxamides A (1) and B (2), including the absolute
configurations of all amino acids, were elucidated by spectroscopic analyses and degradation experiments. The structures are
similar to keramamides F and G, previously isolated from Theonella sp. The analysis of the 16S rDNA sequences obtained from
the metagenomic DNA of D. calyx revealed the presence of Candidatus Entotheonella sp., an unculturable δ-proteobacterium
inhabiting the Theonella genus and implicated in the biosynthesis of bioactive peptides.
calyxamides A and B, as well as the analysis of symbiotic
bacteria in D. calyx.
he lithistid sponges are exceptionally rich sources of
Tstructurally unique and biologically active natural pro-
ducts.1 In particular, the members of the family Theonellidae,
including the Theonella and Discodermia genera, contain potent
cytotoxic macrolides, such as the swinholides2 from the
Theonella genus and discodermolide3 from Discodermia
dissoluta. Strongly bioactive peptides have also been isolated,
such as cyclotheonamide4 and keramamides5 from the
Theonella genus and discodermins6 from the Discodermia
genus. Discodermia calyx collected around Japan contains high
concentrations of calyculins,7 which are the PKS and NRPS
hybrid products that exhibit potent and specific inhibition
against protein phosphatases 1 and 2A. Recently, Jung and co-
workers reported C-21 furanoterpenes, bisindole alkaloids, and
bromohistidine derivatives isolated from a Korean specimen of
D. calyx.8 However, there has been no report regarding the
isolation of polypeptides from D. calyx, despite the presence of
bioactive peptides, such as discodermin6 and discobahamins,9 in
sponges of the same genus. The presence of the abundant and
potent calyculins (calyculin A; 0.15% in wet weight)7 could
mask the presence of other minor cytotoxic compounds.
Therefore, a further detailed investigation of D. calyx, collected
off of Shikine-jima Island, Japan, led to the isolation of the
cytotoxic cyclic peptides calyxamides A and B, which are
structurally related to the keramamides5 isolated from Theonella
sp. Herein, we report the isolation and structural elucidation of
The sponge D. calyx was collected in the ocean near
Shikine-jima Island, Japan. The concentrated MeOH extract
of the frozen sponge (2.5 kg) was successively partitioned
between hexane and H2O, and then the latter layer was
repartitioned between EtOAc and H2O. The EtOAc extract was
subjected to chromatography on a silica gel column, followed
by reversed-phase HPLC on ODS to yield calyxamides A
(1, 3.0 × 10−4 % wet weight) and B (2, 1.0 × 10−4 % wet
weight) as yellow solids.
The molecular formula of calyxamide A (1) was established
1
as C45H61N11O12S by positive ion ESI-TOFMS. The H NMR
spectrum (DMSO-d6) of 1 exhibited α- and amide protons,
thereby implying its peptide nature. In addition, a minor set of
1
resonance signals existed along with the major one in the H
NMR spectrum in CD3OD. An extensive analysis of the 1D and
2D NMR data of 1 (Table 1), including 1H−1H COSY,
HMQC, and HMBC spectra in DMSO-d6, suggested the
presence of a trans-olefin and alanine (Ala), isoleucine (Ile),
and 2,3-diaminopropionic acid (Dpr) residues. Further-
more, there was a modified Ile with a downfield proton (δH =
5.17) that showed an HMBC correlation with the carbonyl
carbon (δC = 198.2), which was consistent with an AKMH
Received: November 20, 2011
Published: January 25, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Note
moiety and the Htrp moiety, to yield O-methylserine and aspartic
acid, respectively. Treatment of 1 with H2O2/NaOH transformed
the AKMH into isoleucine. The O-methylserine, aspartic acid,
and isoleucine residues thus obtained were also determined to
be the L-forms by the chiral-phase GC method, indicating the
L-configuration of all amino acids in 1. Therefore, the complete
structure of calyxamide A was concluded to be 1.
The molecular formula of calyxamide B (2) was established
as C45H61N11O12S, by the positive ion ESI-TOFMS, which was
the same as that of calyxamide A (1). Extensive analysis of the
NMR data of 2 (Table 1), by comparison with those of 1,
confirmed that the planar structure of 2 was the same as that of 1.
The differences between 1 and 2 were found for the 1H and 13C
chemical shifts at the β-NH of Dpr (1, δH = 7.17; 2, δH = 7.69),
the α-proton of Ala (1, δH = 4.36; 2, δH = 4.62), H-13
(1, δH = 5.17; 2, δH = 5.42), H-9 (1, δH = 5.30; 2, δH = 4.73),
C-13 (1, δC = 61.6; 2, δC = 57.5), C-16 (1, δC = 16.5; 2, δC =
14.9), and C-17 (1, δC = 23.7; 2, δC = 27.3). These differences
between 1 and 2 were similar to those between keramamides F
and G. Thus, these data implied that 2 is the diastereomer of 1,
which was confirmed by the stereochemical assignment. The
chiral-phase GC analysis, after the oxidation of 2 with H2O2/
NaOH followed by acid hydrolysis and derivatization, furnished
D-Ile, as expected. In addition, all of the other amino acid re-
sidues in 2 were the L-form, as in the case of 1. Therefore, the
complete structure of calyxamide B was concluded to be 2.
Calyxamides A (1) and B (2) showed moderate cytotoxicity
against P388 murine leukemia cells, with IC50 values of 3.9 and
0.9 μM, respectively. The overall structures are closely related
to those of keramamides F, G, H, J, and K, isolated from the
Okinawan marine sponge Theonella sp.,5 but the calyxamides
are the first cytotoxic cyclic peptides isolated from the Japanese
marine sponge, D. calyx.
The α-ketoamide functionality was originally found in the
cyclotheonamides4 isolated from the Japanese sponge Theonella
sp., collected near Hachijo-jima Island by Fusetani and co-
workers in 1990. Subsequently, the keramamides5 were isolated
from an Okinawan Theonella sp. Although these α-ketoamide-
containing cyclic peptides appeared to be unique secondary
metabolites for the genus Theonella, similar peptides, the
discobahamins from a Discodermia sp., collected from the deep
sea off the Bahamas have been reported.9 The structural
resemblance between the secondary metabolites from Theonella
and Discodermia sponges is not limited to the cyclic peptides,
but also includes some closely related polyketides, such as the
calyculin-related metabolite swinhoeiamide A11 and theopeder-
ins.12,13 These findings suggest the existence of various
chemotypes in the Theonellidae family of sponges and the
possibility of secondary metabolite production by symbiotic
bacteria.14,15 A correlation has been demonstrated between the
presence of filamentous bacteria and the isolation of cyclic
peptides.18 In particular, a filamentous bacterium associated
with Theonella swinhoei has been shown to be the Candidatus
Entotheonella palauensis, producing the cyclic peptide theopa-
lauamide.16,17 On the other hand, only two groups have
independently reported the presence of Entotheonella sp. in the
genus Discodermia.18,19 The presence of the calyxamides in
D. calyx, which are closely related to peptides from Theonella,
raised the possibility that D. calyx also harbors Entotheonella as
symbiotic bacteria, although the bacterial community associated
with D. calyx has remained unexplored. To obtain general
insights into the bacterial community in D. calyx, a microscopic
analysis of the symbiosis was conducted. Consequently we
(3-amino-2-keto-4-methylhexanoic acid) residue. In addition,
the presence of a thiazole ring conjugated to the trans-olefin
was indicated by the HMBC spectroscopic data of 1 [δH = 7.81
(s, 1H); δC = 168.0, 132.4, and 149.9]. The amino acid com-
position, the α-ketoamide functionality, and the thiazole ring
system were reminiscent of those of keramamide F,5b a cyto-
toxic cyclic heptapeptide from the marine sponge Theonella sp.
A comparison of the NMR data with those reported in the
literature readily revealed that 1 had the same partial structure
(Ile-Dpr-Ala-a-b) of the keramamide F skeleton, which contains
modified amino acid residues. However, the remaining two
portions, corresponding to the isoserine and tryptophan
residues of keramamide F, were different. Three aromatic
protons (δH = 6.53, 6.83, and 7.03) and an exchangeable proton
(δH = 10.41) coupled to another aromatic proton (δH = 6.80)
were consistent with a 5-hydroxy-3-substituted indole, which
was supported by the UV absorption maximum at 278 nm. The
HMBC correlations of the methylene protons (δH = 3.02 and
3.21), which were no longer olefinic, indicated the presence of
5-hydroxytryptophan (Htrp),10 in place of the α,β-dehydro-
tryptophan in keramamide F. The last amino acid residue
1
exhibiting characteristic H NMR signals [δH = 1.67 (m, 1H),
1.83 (m, 1H), 2.05 (m, 2H), 6.74 (brs, 1H), and 7.26 (brs,
1H)] was identified as glutamine (Gln), which was connected
to Ile. The HMBC correlation between a formyl proton and the
α-methine carbon (δC 51.2) of Gln indicated the presence of a
formyl group (δH 7.98; δC 161.4) attached to the N-terminus.
Evidence for the amino acid sequence of 1 was provided by the
NOESY and HMBC correlations and established the cyclic
portion of this peptide 1. On the basis of these NMR data, the
gross structure of calyxamide A (1) was established. There was
an unassignable 13C signal at 99.7 ppm in CD3OD, which,
together with the presence of a minor ion peak at m/z 1034
[M + CH3OH + Na]+ in the ESI-TOFMS spectrum, suggested
hemiacetal formation with MeOH at the α-ketoamide portion
in 1, as the minor component.4
A chiral-phase GC analysis of the N-trifluoroacetyl/methyl
ester derivatives of the hydrolysate of 1 clarified that all of the
Ala, Gln, Ile, and Dpr residues in 1 were the L-form. Treatment of
1 with ozone led to the degradation of the (O-methylseryl)thiazole
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Journal of Natural Products
Note
1
Table 1. H and 13C NMR Chemical Shifts of Calyxamides A (1) and B (2) in DMSO-d6
calyxamide A (1)
calyxamide B (2)
calyxamide A (1)
calyxamide B (2)
a
b
a
b
a
b
a
b
position
δC
δH (J in Hz)
δC
δH (J in Hz)
position
δC
δH (J in Hz)
δC
δH (J in Hz)
CHO
Gln
161.4
172.0
7.98, s
161.5
171.4
7.99, s
7
9
168.0
51.9
166.2
54.3
CO
α-NH
α
5.30, dt (6.2,
8.5)
4.73, q (6.8)
9.24, d (6.8)
8.24, d (8.5)
8.22, d (7.9)
c
c
10
11
9.21, d (8.5)
51.2
28.7
4.33, m
51.2
28.8
4.36, m
c
c
73.4
3.58, dd (6.8,
9.6)
73.1
3.89, dd (5.7,
9.1)
β
1.67, m
1.68, m
1.83, m
2.05, m
1.85, m
2.06, m
3.66, dd (5.7,
9.6)
3.95, t (8.5)
γ
31.9
31.9
δ
174.2
174.3
12
13
14
15
59.0
164.6
198.2
61.6
3.26, s
58.9
161.5
197.7
57.5
3.35, s
δ-NH2
6.74, brs
7.26, brs
6.71, brs
7.27, brs
b
Ile
CO
NH
α
171.3
171.2
5.17, dd (2.8,
8.5)
5.42, dd (2.8,
9.6)
7.92, d (8.5)
4.16, t (8.2)
7.92, d (9.1)
4.18, t (7.9)
16
17
18
19
8.58, d (8.5)
2.21, m
8.57, d (9.6)
57.3
37.2
15.9
24.8
57.2
37.3
15.9
24.8
c
c
c
37.4
16.5
23.7
37.5
14.9
27.3
2.49, m
β
1.67, m
1.69, m
c
0.84, d (7.4)
0.74, d (7.4)
1.20, m
γ-CH3
γ-CH2
0.79, m
0.80, d (6.2)
1.05, m
1.13, qui
(7.4)
1.03, m
1.37, m
1.39, m
20
12.3
0.73, t (7.4)
12.2
0.83, t (7.4)
c
δ-CH3
CO
α-NH
α
11.5
0.78, m
11.6
0.78, t (7.4)
Htrp
CO
α-NH
α
171.2
172.2
Dpr
170.9
169.9
c
7.65, d (9.6)
7.97, m
c
8.14, d (6.8)
7.95, m
53.6
30.0
4.78, dt (3.8,
10.5)
53.8
29.5
4.56, dt (3.4,
11.9)
c
c
52.0
41.0
4.30, m
51.9
40.7
4.37, m
β
2.71, m
3.78, m
2.78, m
β
3.02, dd
(11.6, 15.0)
2.96, dd (11.9,
14.7)
3.57, m
c
c
β-NH
7.17, dd (6.2,
9.1)
7.69, t (6.2)
3.21, m
3.30, m
1′-NH
10.41, d (1.7)
6.80, d (1.7)
10.38, d (1.7)
6.78, d (1.7)
Ala
CO
NH
α
175.4
174.7
2′
123.2
110.0
102.7
150.8
123.7
110.7
102.5
150.8
8.34, d (4.0)
8.07, d (6.8)
4.62, t (7.1)
1.30, d (7.4)
3′
c
49.8
17.7
4.36, m
49.1
20.3
4′
6.83, d (2.3)
8.59, brs
6.82, d (2.3)
8.54, s
β
1.24, d (6.8)
5′
a
1
165.3
124.4
132.4
149.9
123.8
165.0
125.1
131.7
149.8
123.0
5′-OH
6′
2
6.83, d (15.3)
7.27, d (15.3)
6.60, d (15.3)
7.15, d (15.3)
111.9
6.53, dd (2.3,
8.5)
111.7
6.53, dd (2.3,
8.5)
3
4
7′
8′
9′
112.2
131.1
128.7
7.03, d (8.5)
112.2
131.3
128.3
7.04, d (8.5)
5
7.81, s
7.73, s
a
b
c
In 500 MHz. In 125 MHz. Overlapped.
Entotheonella sp. from Discodermia dissoluta (GenBank accession
no. AY897123), and the Candidatus Entotheonella palauensis
from Theonella swinhoei was also ranked highly (GenBank
accession no. AF130847, 96% identity). The common existence
of Candidatus Entotheonella sp. in both Theonella and
Discodermia may explain the similarity of the secondary
metabolites in these two genera.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO DIP-1000 digital polarimeter. 1H and 13C
NMR spectra were recorded on a JEOL ECX-500 spectrometer in
Figure 1. Key HMBC and NOESY correlations of calyxamide A in
DMSO-d6.
1
DMSO-d6, CD3OD, and CD3OH. H and 13C NMR chemical shifts
were reported in parts per million and referenced to solvent peaks:
δH = 2.46 and δC = 40.0 ppm for DMSO-d6, δH = 3.29 and δC = 47.8
for both CD3OD and CD3OH. HRMS data were obtained from a
Bruker Daltonics micro TOF-MS.
Animal Material. The marine sponge Discodermia calyx was
collected by hand using scuba at a depth of 10 m in the ocean near
Shikine-jima Island, 150 km south of Tokyo, and was kept frozen until
use. The voucher specimen (S11-001) was deposited at the Laboratory
of Natural Products Chemistry, Graduate School of Pharmaceutical
Sciences, the University of Tokyo. The fresh specimen was minced
identified the presence of filamentous bacteria by light micro-
scopy using separated bacterial cell fractions, as described
previously.16−18 The analysis of the 16S rDNA sequences
obtained by PCR using the metagenomic DNA of D. calyx as the
template revealed the presence of a sequence showing high
similarity to those of δ-proteobacteria. A BLAST search showed
that the sequence was most similar (97% identity of 1479
alignable bp's) to that of the uncultured Candidatus
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Journal of Natural Products
Note
with a knife and suspended in calcium- and magnesium-free artificial
seawater, and the suspension was observed by light microscopy.
Isolation. The MeOH (2.0 L × 1, 0.5 L × 3) extract of the sponge
D. calyx (0.9 kg, wet weight) was partitioned between hexane
(350 mL × 2) and H2O (350 mL). The aqueous layer was further
partitioned between EtOAc (350 mL × 2) and H2O (350 mL). The
EtOAc-soluble material (0.72 g) was subjected to open chromatography
on a silica gel column, eluting with a stepwise gradient of EtOAc (0−90%)
in hexane, and MeOH (15−20%) in CHCl3. The fractions eluted with
CHCl3/MeOH were then separated by reversed-phase HPLC on ODS
[Cosmosil MS-II column ⦶ 10 × 250 mm; flow rate 4.0 mL/min; 30−
100% CH3CN/H2O over 30 min; UV detection at 280 nm] to give
fraction I and calyxamide B (2, 0.9 mg). Fraction I was further purified
by reversed-phase HPLC on ODS [Cosmosil MS-II column ⦶ 10 ×
250 mm; flow rate 4.0 mL/min; 33% CH3CN containing 0.05% TFA;
UV detection at 280 nm] and cholesterol [Cosmosil Cholester, ⦶
10 × 250 mm; flow rate 4.0 mL/min; 33% CH3CN containing 0.05%
TFA; UV detection at 280 nm], to yield calyxamide A (1, 2.7 mg, tR =
10.0 min, 13.0 min). Moreover, additional sponge (1.6 kg) was
extracted and separated in the same manner as above to afford 1
(4.8 mg) and 2 (1.6 mg).
into lysis buffer (8 M urea, 2% sodium dodecyl sulfate, 350 mM NaCl,
50 mM EDTA, 50 mM Tris [pH 7.5]), using 5 mL per g of sponge
tissue, for 1 h at 60 °C with gentle mixing. The lysate was extracted
with an equal volume of phenol/CHCl3/isoamyl alcohol (25:24:1)
and with an equal volume of CHCl3/isoamyl alcohol (24:1). The
DNA was then precipitated by 2.5 volumes of EtOH and 1/10 volume
of 3 M sodium acetate (pH 5.2) and was washed with 70% EtOH
at 4 °C.
PCR Conditions. Amplification of rDNA was performed with the
universal eubacterial primers20 27f and 1492r, using the metagenomic
DNA of D. calyx. The PCR cycling conditions were as follows: initial
denaturation (95 °C for 5 min), followed by 35 cycles of denaturation
(95 °C for 30 s), annealing (55 °C for 30 s), and extension (72 °C for
90 s), with a final extension step (72 °C for 10 min). The PCR
products were ligated into the pT7Blue vector (Novagen) using a
ligation kit (Takara) and were transformed in Escherichia coli DH5α.
Plasmid DNA was isolated by a Wizard Plus SV Minipreps System
(Promega).
Sequencing. DNA sequencing was performed using an ABI
PRISM 3100 genetic analyzer (Applied Biosystems). The M13
universal and reverse primers and the 16S rDNA-specific primer,
514f, were used in the complete sequencing of the 16S rDNA
amplicons. The sequence data were analyzed using NCBI BLAST and
deposited at DDBJ (accession no. AB683979).
Cytotoxicity Test against P388 Cells. P388 murine leukemia
cells were cultured in RPMI 1640 (Wako Chemicals) medium,
supplemented with 10 μg/mL of penicillin/streptomycin (Invitrogen)
and 10% fetal bovine serum (MP Biomedicals), at 37 °C under a
5% CO2 atmosphere. To each well of 96-well microplates, containing
100 μL of 1 × 104 cells/mL tumor cell suspension, was added 100 μL
of test solution (samples were dissolved in DMSO), and the plates were
incubated for 4 days. After the addition of 50 μL of 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide solution (1 mg/mL)
to each well, the plates were incubated for 4 h under the same
conditions. The mixtures were centrifuged, and the supernatants were
removed. The precipitates thus obtained were dissolved in DMSO,
and the absorbance at 570 nm was measured with a microplate reader.
Calyxamide A (1): yellow solid; [α]27 −15.4 (c 0.62, MeOH);
D
1
UVmax (MeOH) 278 nm; H and 13C NMR (Table 1); ESI-TOFMS
m/z 1002.4144 [M + Na]+ (calcd for C45H61N11NaO12S, 1002.4114).
Calyxamide B (2): yellow solid; [α]32 −25.3 (c 0.28, MeOH);
D
UVmax (MeOH) 278 nm; H and 13C NMR (Table 1); ESI-TOFMS
1
m/z 1002.4154 [M + Na]+ (calcd for C45H61N11NaO12S, 1002.4114).
Amino Acid Analysis by Chiral-Phase GC. Calyxamide A or B
(200 μg each) was hydrolyzed with 6 M HCl (500 μL) at 110 °C for
24 h. The reaction mixture was treated with 5−10% HCl/MeOH (500 μL)
at 100 °C for 30 min and was then treated with trifluoroacetic
anhydride (TFAA)/CH2Cl2 (1:1, 500 μL) at 100 °C for 5 min. The
chiral-phase GC analysis of the N-trifluoroacetyl (TFA)/methyl ester
derivatives was performed using a CP-Chirasil-D-Val column (Alltech,
0.25 mm × 25 m; N2 as the carrier gas; program rate 50−200 at 4 °C/
min) and showed peaks at tR = 4.3, 8.1, 17.6, and 26.6 min. Standard
amino acids were also converted to the TFA/Me derivatives by the
same procedure. Retention times (min) were as follows: L-Ala (4.3), D-
Ala (5.0), L-Ile (8.1), D-Ile (8.8), L-allo-Ile (7.6), D-allo-Ile (8.4), L-Gln
(17.6), D-Gln (18.4), L-Dpr (26.6), D-Dpr (27.1). Thus, the presence
of L-Ala, L-Ile, L-Gln, and L-Dpr was confirmed.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectroscopic data for 1 and 2. This material is available
Determination of the Configurations of (O-Methylseryl)-
thiazole and 5-Hydroxytryptophan. A stream of ozone in oxygen
was bubbled through a cooled solution of calyxamide A or B (200 μg
each) in MeOH (3 mL) at −78 °C for about 15 min. The reaction was
quenched with 30% H2O2 (15 drops) and allowed to warm to room
temperature (rt). After 1 h, the solvent was removed under nitrogen.
The reaction mixture was subjected to hydrolysis and TFA/Me
derivatization. The chiral-phase GC analysis of the resulting hydro-
lysate was performed as above and showed peaks at tR = 4.3, 6.9, 8.1,
12.8, 17.6, and 26.6 min, which established the presence of L-O-
methylserine and L-aspartic acid. Retention times (min) were as
follows: L-O-methylserine (6.8), D-O-methylserine (7.3), L-Asp (12.8),
D-Asp (13.2).
Determination of the Configuration of the C11−C14 Moiety.
Calyxamide A or B (200 μg each) in 5% NaOH (500 μL) was treated
with 30% H2O2 (100 μL) at 65 °C for 40 min. After cooling to rt
overnight, the reaction mixture was subjected to hydrolysis, followed
by TFA/Me derivatization. The resulting hydrolysate was subjected to
the chiral-phase GC analysis as above. The hydrolysate derived from 1
showed peaks at tR = 4.3, 8.1, 17.6, and 26.6 min. Only the L-form of
Ile (tR = 8.1 min) was observed, and the peak area for Ile was
increased, as compared to that derived from the normal hydrolysate
from 1. In contrast, the resulting hydrolysate of 2 showed peaks at tR =
4.3, 8.1, 8.8, 17.6, and 26.6 min. The D-form of Ile (tR = 8.8 min) was
detected in addition to the L-form of Ile (tR = 8.1 min).
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: wakimoto@mol.f.u-tokyo.ac.jp, abei@mol.f.u-tokyo.ac.
jp. Phone: +81-3-3818-2532. Fax: +81-3-5841-4744.
ACKNOWLEDGMENTS
■
We thank Dr. K. Takada and Prof. S. Matsunaga (Graduate
School of Agricultural and Life Sciences, The University of
Tokyo) for the generous gift of the specific primers for 16S
rDNA. We also thank Professor J. Piel (University of Bonn) for
insightful suggestions. This work was partly supported by The
Mitsubishi Foundation, The University of Tokyo Global COE
Program (Center for Medical System Innovation), and a Grant-
in-Aid for Young Scientists (B) from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan.
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